1. Field of the Invention
The invention relates to a method for identifying a Mycobacterium species responsible for a mycobacterial infection in human or animal. The invention further relates to diagnostic kits for use in the method.
2. Description of the Related Art
The genus Mycobacterium which contains about 50 species, is responsible for a number of human and animal diseases which are known collectively as the mycobacterioses. The best known of these in humans are leprosy, caused by M. leprae, which affects more than ten million people worldwide, and tuberculosis, usually caused by M. tuberculosis, at least ten million new cases of which occur each year. Most other mycobacteria normally occur only as environmental saprophytes but can also cause opportunist diseases. This happens usually, but not only, in the case of people who have problems with their immune system, such as AIDS patients and people undergoing immunosuppression. These opportunist types comprise the slow-growing species M. avium, and the closely related M. intracellulare and M. scrofulaceum (often referred to together as MAIS complex), M. kansasi, M. marinum and M. ulcerans, and the fast-growing species M. chelonae and M. fortuitum. Although once rare, the incidence of opportunist mycobacterial diseases and tuberculosis shows a parallel increase in the western world with the incidence of AIDS. In addition there is limited but increasing evidence that mycobacteria or antigens thereof play a direct or indirect part in the etiology of a plurality of other diseases such as sarcoidosis and Crohn""s disease and different auto-immune diseases such as auto-immune dermatitis, rheumatoid arthritis and diabetes. This could be attributed to a structural mimicry between epitopes of mycobacteria and those of the host.
The cell walls of mycobacteria are very complex and contain many lipids, some with structures unique to the genus. These structures comprise mycolinic acids and esters, peptido-glycolipde, arabino-galactane and lipo-arabino-manane. The lipid-rich mycobacterial cell walls are responsible for the characterizing coloring properties of the mycobacteria. They also enable mycobacteria to counter an attack by the immune system of the host. A number of species, once taken up into macrophages, are capable of surrounding themselves with a thick layer of secreted lipids.
Many different components of the mycobacteria begin an interaction with the immune system. These components comprise protein and hydrocarbon antigens, which can either be actively secreted by the mycobacteria or can form part of the cell wall or cell membrane. In addition they may be present in the cytoplasm, for instance in the cytoplasmic matrix, ribosomes and enzymes. Mycobacteria also possess immuno-modulating components such as immunosuppressing compounds and adjuvants. Consequently, a single mycobacterial species can induce a large variety of immune responses in different forms and with diverse specificities. It is therefore difficult to distinguish immune responses against species-specific components from cross reactions. For this reason it has therefore been found difficult to derive protein antigens suitable for the detection of species-specific humoral responses as a basis for a very sensitive and specific sero-diagnostic test for tuberculosis. Because the mycobacteria occur a great deal in the environment, human serum nearly always contains anti-mycobacterial antibodies.
In view of the problems with the specificity of protein antigens, a number of researchers, including the present inventors, have focused their attention on species-specific glycolipid antigens for the detection of specific humoral immune responses. Although the immune reactivity against mycobacteria is of the cell-mediated type and the humoral immune responses probably play a minor part in the total effector mechanism of mycobacterial immunity and immunopathology, studies in the antibody response to immuno-dominant mycobacterial cross-reactive antigen components (referred to hereinafter as xe2x80x9cIm-CRACxe2x80x9d) could shed light on the varying capability of the host to recognize different mycobacterial antigens. They could therefore provide indirect information relating to the nature of the immune recognition of, and response to, a specific mycobacterial pathogen.
It has now been found that the clinical manifestation of mycobacterial diseases appears to be related to the varying capability of an individual host to produce a humoral response to different mycobacterial immuno-cross-reactive antigen components (Im-CRAC). Each mycobacterial infection generates its own specific antibody response to a number of specified antigens. Analysis of the antibody-response by means of immunoblotting has demonstrated that the immuno-dominant Im-CRAC vary in accordance with the immunopathological manifestation of the mycobacterial diseases. It has been found that the sera of individuals which are infected with different Mycobacterium species cause different and distinguishing band patterns on immunoblots of mycobacterial antigens.
This discovery forms the basis of the present invention, whereby a method is provided for identifying a Mycobacterium species responsible for a mycobacterial infection in human or animal, comprising the steps of:
(a) selecting a suitable mycobacterial species and strain;
(b) preparing an antigen preparation comprising at least one mycobacterial antigen;
(c) binding the antigen, respectively the antigen preparation to a suitable carrier;
(d) causing the binding antigen to react with antibodies from serum of an individual infected with a Mycobacterium species;
(e) making visible antigen-antibody reactions for a suitable antibody (sub-)class; and
(f) identifying the responsible Mycobacterium species on the basis of the reactions which are made visible.
In preference, the antigen preparation is separated by electrophoresis prior to step (c) and the carrier is a membrane to which the antigen is bound by means of electroblotting. This process is called Western blotting.
The invention also provides a method for identifying a Mycobacterium species responsible for a mycobacterial infection in a patient by testing a sample comprising antibodies from the patient, the method includes the steps of:
(a) providing an antigenic preparation of a Mycobacterium species, wherein the antigen preparation comprises two or more immuno-cross-reactive antigen components (ImCRACs); wherein the ImCRACs are bound to a solid carrier to form carrier-bound ImCRACs;
(b) contacting the carrier-bound ImCRACs with the sample under conditions suitable for antibody-antigen binding, to provide a set of carrier-bound antibody-ImCRACs; and
(c) detecting the set of carrier-bound antibody-ImCRACs; the set of carrier-bound antibody-ImCRACs being characteristic of the Aycobacterium species.
The invention further provides a method for detecting or identifying an antibody reactive with a Mycobacterium species in a sample. The method includes the steps of:
(a) providing a solid phase carrier having bound thereto an antigen preparation from a culture of the Mycobacterium species, wherein the antigen preparation comprises two or more immuno-cross-reactive antigen components (ImCRACs), forming carrier-bound ImCRACs;
(b) contacting the carrier-bound ImCRACs with the sample under conditions suitable for binding of antibodies in the sample to the carrier-bound ImCRACs to provide a set of carrier-bound antibody-ImCRACs; and
(c) detecting the set of carrier-bound antibody-ImCRACs; wherein the binding of the carrier-bound ImCRACs to antibodies of the sample exhibits a set of antibody-ImCRACs characteristic of the Mycobacterium species.
The invention yet further provides a diagnostic kit for the identification of antibodies elicited by a Mycobacterium species. The kit includes: (i) an antigenic preparation of a Mycobacterium species, wherein the antigen preparation comprises two or more immuno-cross-reactive antigen components (ImCRACs). The ImCRACs are bound to a solid carrier; and binding of the carrier-bound ImCRACs to antibodies reactive with the ImCRACs exhibits a set of antibody-ImCRACs characteristic of particular Mycobacterium species; and (ii) an antibody-enzyme conjugate directed against at least one antibody of an isotype selected from the group consisting of IgG, IgM and IgA.